Neurotransmitter release is initiated by the influx of calcium through voltage-dependent calcium channels within 200 us of the arrival of the action potential at synaptic terminals. Ca2+ currents and synaptic transmission can be inhibited by transmitters acting through the binding of Gbeta subunit of trimeric G proteins to the intracellular loop LI-II of N- and P/Q-type Ca2+ channels. The molecular mechanisms, however, responsible for the targeted modulation of presynaptic Ca2+ channels by G proteins are unclear. We have obtained preliminary evidence that a trimeric Go-alpha-beta-gamma complex binds to the presynaptic protein syntaxin, which in turn serves as the G-protein docking site on the intracellular LII-III loop of presynaptic Ca2+ channels. After activation with GTPgammaS, the G-beta subunit is released from the G protein-syntaxin-Ca2+ channel LII-III loop complex and binds to the intracellular LI-II loop, where G protein modification takes place. Our biochemical results support the notion that the sensitivity to modulation by G proteins requires the association of the presynaptic Ca2+ channels with syntaxin at nerve terminals. We are currently testing one of our proposed mechanisms for G protein modulation of neuronal Ca2+ channels in biochemical and electrophysiological systems. Associations between proteins present on transmitter-containing vesicles and on the presynaptic membrane are thought to underlie docking and fusion of synaptic vesicles with the plasma membrane, which are obligate steps in calcium-dependent neurotransmission. Most of the presynaptic proteins involved in synaptic vesicle docking and fusion processes and their interactions have been identified with biochemical techniques and binding assays. Low abundance proteins and weak and transient interactions, probably the norm for large native synaptic protein complexes, would not be detected with these conventional methods. The yeast two-hybrid cloning technique offers the possibility of identifying additional proteins involved in membrane trafficking and synaptic vesicle fusion. Our preliminary search for such proteins using syntaxin as bait has resulted in the isolation of several proteins including SNAP-25 and alpha-SNAP which had been reported to bind syntaxin in vitro biochemical assays. These findings validate the yeast two-hybrid approach. We have also discovered a new isoform of rat beta-SNAP, and partially characterized it. Northern blots revealed that its mRNA, 5 kb in length, is only detectable in the brain. In vitro biochemical studies show that the recombinant His-tagged protein of this new isoform of the beta-SNAP gene is capable of binding to syntaxin in a calcium- dependent manner. Thus, our data raise the possibility that this brain- specific beta-SNAP may play a role in regulating fusion of synaptic vesicles upon Ca2+ influx through presynaptic calcium channels.